Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

Abstract

Background and Aim HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired. Methods Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis. Results The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001). Conclusion Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.

A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis

“…33 We demonstrated HCC development in the cirrhotic liver is accompanied by impaired autophagy and increased EV shedding (microvesicle and exosomes). [34][35][36][37] This data is consistent with reports from other researchers indicating that cancer cells secrete significantly higher amounts of exosomes and microvesicles than normal cells, which could be harnessed as a minimally invasive biomarker for cancer diagnosis. 38,39 However, isolating and quantifying organ-specific exosomes has created a significant hurdle to establishing causal relationships between EV biology and underlying disease.…”

“…The performance of HCC exosome quantification by F-NTA in the same set of samples was compared with the magnetic bead assay described by our group previously. 37 The immunomagnetic beads were prepared by mixing streptavidin-conjugated magnetic beads with biotinylated CD63 antibodies, followed by blocking and incubation directly with the serum for EV capture. This assay principle is that serum exosomes immobilized onto beads reacted either with PE-labeled AFP or PE-labeled GPC3 or in a combination of both antibodies ( Figure 9A ).…”

“…The performance of HCC exosome quantification by F-NTA in the same set of samples was compared with the magnetic bead assay described by our group previously. 37 The immunomagnetic beads were prepared by mixing streptavidinconjugated magnetic beads with biotinylated CD63 antibodies, followed by blocking and incubation directly with the https://doi.org/10.2147/JHC.S423043…”

Computational discovery of co-expressed antigens as dual targeting candidates for cancer therapy through bulk, single-cell, and spatial transcriptomics

“…2C). Considering the prevalence of GPC3 as a biomarker and therapeutic target in HCC [32,33], its presence in the top most candidate genes was anticipated, confirming the validity of our approach. However, to ensure the specificity of markers for minimizing therapy-related toxicity, the expression of most favorable HCC marker genes was checked in HCC samples as well as vital organs (Fig.…”

Extracellular Vesicles as Therapeutic and Diagnostic Tools for Chronic Liver Diseases

“…The development of HCC is linked to compromised autophagy, resulting in the increased production of extracellular vesicles, such as exosomes and microvesicles [56,57]. Recent studies revealed complex associations between exosome formation and autophagy, where autophagy machinery can either boost or hinder EV secretion [58]. The modulation of autophagy can significantly influence both the quantity and content of EVs, consequently playing a pivotal role in determining whether autophagy modulators have pro-tumorigenic or anticancer effects.…”